Prostate cancer is the most common cause of cancer and second leading cause of cancer death for men in the United States. Localized disease is potentially curable with radiation or surgery; however, advanced disease remains incurable. Depletion of gonadal testosterone is the upfront standard therapy for advanced disease. Unfortunately, metastatic disease almost always recurs as castration-resistant prostate cancer (CRPC), which is generally the lethal form of this disease. Despite depletion of gonadal testosterone, CRPC remains driven by intratumoral synthesis of androgens. The survival benefit conferred by abiraterone acetate, which blocks CYP17A1, is the best evidence for the necessity of androgen synthesis in the progression of CRPC. Initial or acquired resistance to abiraterone acetate, which was approved by the United States Food and Drug Administration in April 2011 for the treatment of CRPC, has now become the next hurdle to overcome. Recent studies have shown that synthesis of dihydrotestosterone (DHT), the most potent androgen that drives CRPC progression, unexpectedly bypasses testosterone. This finding alters the landscape of potential drug targets and suggests that 3-hydroxysteroid dehydrogenase/isomerase (3HSD) is a potentially important and viable pharmacologic target. The overarching hypothesis of this proposal is that effective pharmacologic inhibition of 3HSD will be an effective treatment for CRPC and tumors that are resistant to abiraterone acetate. In Aim 1, the role and requirement of 3HSD1 and 3HSD2 expression for the development and progression of CRPC will be defined. In Aim 2, the effect(s) of abiraterone acetate on the conversion from dehydroepiandrosterone to androstenedione by 3HSD1 and 3HSD2 will be characterized. In Aim 3, a series of steroidal azoles will be synthesized and candidate inhibitors of recombinant 3HSD1 and 3HSD2 will be identified, which also inhibit CYP17A1. The ultimate anticipated benefit of this proposal is the development of new and improved approaches to the treatment of men with CRPC.